{"files"=>["https://ndownloader.figshare.com/files/1711689"], "description"=>"<p>(<b>A</b>) Features of a new binary yeast-<i>Escherichia</i>-<i>Agrobacterium</i> shuttle vector, pKO1B. (<b>B</b>) Building of gene-deletion cassettes in pKO1B by yeast recombinational cloning. The 5′ and 3′ flanking fragments of the targeted genes were separately amplified from genomic DNA with primers 5f/5r and 3f/3r. Primers 5r and 3f have 5′ tails homologous to the <i>SUR</i> cassette, whereas those for 5f and 3r are homologous to the vector. The two flanks were cotransformed into yeast along with the <i>SUR</i> cassette and gapped pKO1B. Homologous recombination created the circular knockout vector, and the final knockout vector was subsequently transformed into <i>A. tumefaciens</i>. (<b>C</b>) Deletion of the targeted gene. The gene-deletion cassette was transformed into the fungal cells via ATMT. Homologous recombination created three types of transformants: null mutants, ectopic insertion transformants, and null and ectopic insertion mutants. The <i>GFP</i> gene was discarded in the null mutants. Primers p1/p2 or p3/p4 were used to identify the unique recombinational DNA fragment, indicating a knockout event. (<b>D</b>) The transformants are screened for GFP fluorescence under a microscope. Putative null mutants do have not GFP fluorescence, but ectopic transformants do. (<b>E</b>) The transformants are screened by double PCR for the targeted gene using the <i>β-tubulin</i> gene as a positive control. The wild-type strain or ectopic transformants produced a characteristic band, indicating the targeted gene, while the null mutants did not. (<b>F</b>) The transformants are screened by PCR for a unique recombinational DNA fragment marked as a knockout event. The null mutants have a 1.2–2.0 kb band on an electrophoretic gel, while the wild-type strain and the ectopic transformants do not.</p>", "links"=>[], "tags"=>["104 Zn 2Cys TF genes", "GPF", "gene expression network", "Zn 2Cys genes", "rice blast fungus Magnaporthe oryzae", "9 abiotic stresses", "cnf", "TF genes", "1b", "Rice Blast Fungus", "Zn 2Cys Transcription Factors"], "article_id"=>1199321, "categories"=>["Biological Sciences"], "users"=>["Jianping Lu", "Huijuan Cao", "Lilin Zhang", "Pengyun Huang", "Fucheng Lin"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1004432.g001", "stats"=>{"downloads"=>0, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Overview_of_the_high_throughput_gene_knockout_system_in_fungus_/1199321", "title"=>"Overview of the high-throughput gene knockout system in fungus.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-10-09 03:35:23"}

{"files"=>["https://ndownloader.figshare.com/files/1711769", "https://ndownloader.figshare.com/files/1711770", "https://ndownloader.figshare.com/files/1711771", "https://ndownloader.figshare.com/files/1711772", "https://ndownloader.figshare.com/files/1711773"], "description"=>"<div><p>Because of great challenges and workload in deleting genes on a large scale, the functions of most genes in pathogenic fungi are still unclear. In this study, we developed a high-throughput gene knockout system using a novel yeast-<i>Escherichia</i>-<i>Agrobacterium</i> shuttle vector, pKO1B, in the rice blast fungus <i>Magnaporthe oryzae</i>. Using this method, we deleted 104 fungal-specific Zn<sub>2</sub>Cys<sub>6</sub> transcription factor (TF) genes in <i>M. oryzae</i>. We then analyzed the phenotypes of these mutants with regard to growth, asexual and infection-related development, pathogenesis, and 9 abiotic stresses. The resulting data provide new insights into how this rice pathogen of global significance regulates important traits in the infection cycle through Zn<sub>2</sub>Cys<sub>6</sub>TF genes. A large variation in biological functions of Zn<sub>2</sub>Cys<sub>6</sub>TF genes was observed under the conditions tested. Sixty-one of 104 Zn<sub>2</sub>Cys<sub>6</sub> TF genes were found to be required for fungal development. In-depth analysis of TF genes revealed that TF genes involved in pathogenicity frequently tend to function in multiple development stages, and disclosed many highly conserved but unidentified functional TF genes of importance in the fungal kingdom. We further found that the virulence-required TF genes <i>GPF1</i> and <i>CNF2</i> have similar regulation mechanisms in the gene expression involved in pathogenicity. These experimental validations clearly demonstrated the value of a high-throughput gene knockout system in understanding the biological functions of genes on a genome scale in fungi, and provided a solid foundation for elucidating the gene expression network that regulates the development and pathogenicity of <i>M. oryzae</i>.</p></div>", "links"=>[], "tags"=>["104 Zn 2Cys TF genes", "GPF", "gene expression network", "Zn 2Cys genes", "rice blast fungus Magnaporthe oryzae", "9 abiotic stresses", "cnf", "TF genes", "1b", "Rice Blast Fungus", "Zn 2Cys Transcription Factors"], "article_id"=>1199386, "categories"=>["Biological Sciences"], "users"=>["Jianping Lu", "Huijuan Cao", "Lilin Zhang", "Pengyun Huang", "Fucheng Lin"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1004432.s001", "https://dx.doi.org/10.1371/journal.ppat.1004432.s002", "https://dx.doi.org/10.1371/journal.ppat.1004432.s003", "https://dx.doi.org/10.1371/journal.ppat.1004432.s004", "https://dx.doi.org/10.1371/journal.ppat.1004432.s005"], "stats"=>{"downloads"=>20, "page_views"=>40, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Systematic_Analysis_of_Zn_2_Cys_6_Transcription_Factors_Required_for_Development_and_Pathogenicity_by_High_Throughput_Gene_Knockout_in_the_Rice_Blast_Fungus/1199386", "title"=>"Systematic Analysis of Zn<sub>2</sub>Cys<sub>6</sub> Transcription Factors Required for Development and Pathogenicity by High-Throughput Gene Knockout in the Rice Blast Fungus", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2014-10-09 03:35:23"}